NanoTPOT: Enhanced Sample Preparation for Quantitative Nanoproteomic Analysis

With
the ever-growing need for protein-level understanding in pathological
research, proteomics researchers thrive to examine detailed proteome
dynamics using crucial, yet often limited, primary and clinical samples.
Aside from mass spectrometer instrumentation advancement, a single-tube-based
high-throughput sample processing workflow is imperative to ensure
sensitive, quantitative, and reproducible protein analysis for these
increasingly sophisticated studies. Leveraging the benefits of an
acid-cleavable detergent, RapiGest SF Surfactant (Waters Corporation),
we developed and optimized a nanoproteomic workflow that we termed
Nanogram TMT Processing in One Tube (NanoTPOT). Through the assessment
of proteolytic digestion, tandem mass tag (TMT) labeling, online and
offline fractionation strategies, our optimized workflow effectively
eliminated the need for sample desalting and enabled compatible sample
processing for mass spectrometry analysis. We further applied the
NanoTPOT workflow to examine cellular response to stress caused by
dithiothreitol in HeLa cells, where we identified and quantified 6935
and 5474 proteins in TMT 10-plex experiments with one microgram of
lysate protein and 2000 sorted HeLa cells (roughly half microgram
lysate protein) in each channel, respectively. Our workflow has been
proven to be an effective alternative for current nanoproteomic sample
processing to minimize sample loss in biological and clinical applications.